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Modified Nucleosides: Structure and Medical Applications
Nucleosides, like nucleotides, contain a ribose sugar (deoxyribose or ribose) and nitrogenous base. Unlike nucleotides however, nucleosides have no associated phosphate groups. Nucleosides can be considered the non-phosphorylated precursor molecules of nucleotides. Nucleosides are an integral part of genetic research as they have a broad range of applications in biotechnology, argochemistry and medicine but before we can truly appreciate nucleosides and their many applications, it is imperative to have a basic understanding of DNA structure (1). Nucleotide, Nucleoside and DNA structure DNA, the double-stranded, helical macromolecule that is considered by many to be the blueprint of life, is a nucleic acid polymer made up of nucleotides. Nucleotides are composed of a ribose sugar, one of 5 nitrogenous bases (adenine, cytosine, guanine, uracil and thymine), and at least 1 phosphate group. Nucleotides that are eventually incorporated into DNA have deoxyribose, as opposed to ribose, as their ribose sugar and 1 of 4 possible nitrogenous bases: adenine, cytosine, guanine, and thymine. Deoxyribose differs structurally from ribose because while ribose has a hydroxyl group and a single hydrogen atom attached to its 2' carbon, deoxyribose has only 2 hydrogen atoms bound at its 2' site (when numbering the carbons within a nucleotide, it is customary to label the carbon bound to the nitrogenous base 1', and to continue numbering in a clockwise direction) (1). Individual nucleotides become nucleic acid polymers (like DNA) through the formation of phosphodiester bonds between the sugar group of a nucleotide that has already been incorporated into the nucleic acid chain and the phosphate group of an incoming triphosphate nucleotide. This bond is formed when the 3' hydroxyl group of a nucleotide ribose sugar nucleophilically attacks the phosphorous atom of the phosphate group of a nucleotide triphosphate molecule; this causes the expulsion of a pyrophosphate (2 phosphate groups)(1). Because of the chemistry of phosphodiester bond formation, DNA synthesis can only occur in a 5' to 3' direction. In other words, because it is the 3' hydroxyl group of the preceeding nucleotide that functions in bond formation (attacking the phosphate group attacked to the 5' carbon of the incoming nucleotide), the 5' end of this preceeding nucleotide will be at the begining of this forming nucleotide sequence while the 3' end of the last nucleotide will be at the terminus. It is important to note here that only triphosphate nucleotides (nucleotides with 3 associated phosphate groups) can be incorporated into a growing DNA strand. These phosphodiester bonds create the sugar and phosphate backbone of DNA, but what causes DNA's double stranded, helical nature? The answer is hydrogen bonding between complementary nitrogen bases. Adenine (a purine base) always binds thymine (a pyrimidine base) with 2 hydrogen bonds while cytosine (a pyrimidine base) always binds guanine (a purine base) with 3 hydrogen bonds.. You will notice that a purine base always pairs with a pyrimidine base. This is because purine bases, which are composed of 2 heterocylic aromatic rings as opposed to 1 (like pyrimidines), are considerably bulkier than pyrimidine bases, meaning that if purines were to pair with purines and pyrimidines were to pair with pyrimidines, the width of the DNA double helix would be inconsistent and the molecule as a whole would be less stable. It should also be noted that because 3 rather than 2 hydrogen bonds connect cytosine and guanine, the bond between these 2 nucleotides is considerably stronger than the bond between adenine and thymine (1). While a nucleotide is within a nucleic acid polymer, it is only associated with 1 phosphate group, but single nucleotides can be associated with 3, 2, 1 or even no phosphate groups (in this latter instance, a hydroxyl group would be present in place of a phosphate group). These nucleotides are termed triphosphate, diphosphate, monophosphate and nucleosides, respectively. A nucleotide can acquire a phosphate group through the enzymatic process of phosphorylation; phosphate groups are removed through hydrolysis. Nucleosides can be phosphorylated within a cell to become mono-, di- and eventually triphopsphate nucleotides, but it is in their non-phosphorylated state that they are of medical and argricultural importance as only nucleosides, not nucleotides, can enter a eukaryotic cell. Nucleosides in Medicine Modified nucleosides have been used successfully in anti-cancer and antiviral applications (1,2). Of particular interest is the role modified nucleosides play in antiretroviral (ARV) therapy (HIV treatment). Nucleoside derivatives used in ARV therapy are typically termed nucleoside reverse transcriptase inhibitors (NRTI). Reverse transcriptase is an enzyme ubiquitously present in the retrovirus viral family (a group of viruses that includes HIV-1 and HIV-2). This enzyme converts the virus's single stranded RNA genome into a double stranded DNA helix capable of integrating into the host genome, a process that first involves the reverse transcription of the viral RNA genome and then DNA replication along the newly synthesized DNA strand (Video). NRTI's terminate chain elongation during DNA synthesis thus preventing the integration of proviral DNA into the human genome (2). Method of NRTI Action Though NRTI's are structurally very similar to natural nucleosides, they will typically have 1 important chemical difference. These differences typically fall into 1 of 2 categories: modified sugar or modified base. NRTI's that have modified deoxyribose groups will typically have a group other than hydroxyl group at their 3' site (as mentioned earlier, this hydroxyl group is essential to phosphodiester bonding) while NRTI's with modified bases will have nitrogenous bases incapable of forming hydrogen bonds with their complementary base, thus preventing the formation of a DNA double helix (3). While the host's DNA polymerase will typically abstain from using these modified nucleoside products, reverse transcriptase will eagerly try to incorporate them into its growing DNA chain, causing the cessation of DNA synthesis (unless it has mutated to recognize these NRTI products). It is important to note that the "active moiety for all NRTIs is an intracellular 5'-triphosphate compound (2), meaning that these modified nucleosides require intracellular phosphorylation by host mechanisms to become a triphosphate nucleotide analogs before they can exhibit their antiviral effects. In this sense, NRTIs are "prodrugs", meaning that they are the precursor molecules used in the synthesis of an active drug moiety. Why then are modified triphosphate analogs not used in ARV therapy? As mentioned earlier, only nucleosides will enter intact eukaryotic cells. References Print 1. Buckingham, Lela. "Fundamentals of Nucleic Acid Biochemistry." ''Molecular Diagnostics ''. Philadelphia: 2012. Internet 2. Pharmacology of nucleoside reverse transcriptase inhibitors 3. Nucleoside Chemistry